The present invention provides a simple, rapid procedure for characterizing sperm using flow cytometry measuring techniques coupled with staining by various dyes.
Characterizing motility, viability, and sperm morphology can provide useful information not only with respect to reproduction but also as an early warning monitor of excessive invasion by dangerous agents (including radiation) into the body. The mammalian testis is a target organ for chemical toxins in the work place and environment. In addition, radiation from various natural and man made sources can cause degenerative effects. The adverse effects can include a reduction in the number of testicular spermatids, secondary spermatocytes, and sperm--and in even more dramatic cases, a loss of spermatogonia (N.Engl. J. Med. 298:234(1978); Lancet 2:1259 (1977)). In the area of cancer chemotherapy, numerous studies have demonstrated significant, acute and long term gonadal dysfunction in patients receiving chemotherapy, although recovery has been found possible. Recent studies (Science 215:643 (1982) have suggested that effects on reproduction and chromosomes may be the best indicators of exposure to chemicals.
The present invention provides a means whereby sperm may be characterized and thereby act as an indicator of invasions into the body by chemicals, diseases, etc.; as well as a rapid accurate method which may easily be applied in fertility clinics for diagnosis, counseling and prognosis purposes. The inventive method can also be applied for screening persons who may be in contact with excessive radiation as a medical, diagnostic tool e.g. uranium miners who may be in contact with very low level radiation for extended periods of time, making detection by other means impossible or extremely expensive.
Analysis of the effects of gonadal dysfunction by light microscopy, now presently employed, is semiquantitative at best, and is quite subjective and time consuming. Furthermore, conventional staining procedures cannot easily distinguish between various cell types e.g. lymphocytes versus immature spermatids, if this can be accomplished at all. Furthermore, it is difficult, if not practically impossible, to distinguish variations of chromatin condensation by light microscopy usually employed in screening laboratories.
The present invention provides flow cytometry (FCM) methods with significant advantages over the usually applied prior art methods, for the analysis of semen (and testicular biopsies) including: (1) rapidity of measurement (1,000 cells per second); (2) unbiased selection of cells for measurement; (3) simultaneous multi-parameter measurements per cells; (4) objective criteria for classification defined by machine specifications; (5) statistical strengths; and (6) ease of classifying and differentially counting different cell types.
Flow cytometry has been applied in the analysis of sperm precursor cells (Cohn et al N.Engl. J. Med. 298: 234 (1978); DNA content of mature sperm (Gledhill et al in: Flow Cytometry and Sorting, Melamed et al (Eds.), John Wiley & Sons, N.Y. page 471 (1979)) and variations in sperm head shapes (Benaron et al Cytometry 2:344 (1982)) as dosimeters of mutagenic chemical exposure. Similarly, fine needle biopsies of human testis has been studied by flow cytometry of DNA content (Thouid et al Acta Pathol. Microbiol. Scand. 275: 175 (1981)). However no one has previously applied flow cytometry as in the present invention to characterize sperm.